The proposed research is designed to develop and test a novel mouse model for in vivo target validation studies relevant to neurodegenerative diseases. This model would permit regulatable silencing of disease- relevant genes via RNA interference (RNAi). The development of safe and effective therapies for human disease requires extensive preclinical data on the effects of altered expression and/or activity of specific gene products on disease pathogenesis in experimentally tractable model systems. In recent years, RNAi techniques have greatly expanded the repertoire of reverse genetic approaches available for studies of potential therapeutic targets in vivo. RNAi is a powerful tool for experimental manipulation of gene expression and can be used to assess the impact of inhibiting target gene products on the initiation and maintenance of disease in mouse models. The primary goal of this project is to generate a flexible system that permits regulatable RNAi-mediated gene silencing in vivo and to use this system to evaluate the therapeutic potential of gene knockdown approaches for the treatment of dominantly inherited amyotrophic lateral sclerosis (ALS). Approximately 20% of familial ALS (FALS) cases are caused by mutations in Cu, Zn superoxide dismutase (SOD1). Overwhelming evidence implicates novel toxic function(s) of the mutant protein as the cause of disease;for this reason, mutant SOD1 itself is a key therapeutic target for familial FALS. The regulatable RNAi vector generated in the first part of this project will be used to establish transgenic mice in which mutant SOD1 expression can be shut down upon treatment of animals with doxycycline. These experiments will assess whether inducing RNAi-mediated silencing of mutant SOD1 in the early stages of pathogenesis will slow the progression of disease. Earlier studies have demonstrated that constitutive expression, beginning prenatally, of small RNAs targeting mutant SOD1 could ameliorate disease in an ALS mouse model. To date, however, there are no mouse models of ALS that permit temporal regulation of mutant SOD1 protein levels. Given the uniformly rapid progression of ALS in affected individuals, it is critical to determine whether a reduction of mutant protein levels would be beneficial if achieved at the onset of disease symptoms. The proposed experiments are designed directly to determine at what point, during the life of an animal engineered to develop motor neuron disease, a reduction in the amount of mutant SOD1 protein will provide a significant therapeutic benefit. The results of this study will aid in establishing an appropriate window for applying agents aimed at reducing mutant protein levels in mutant SOD1-mediated ALS and will provide key data regarding the therapeutic potential of RNAi-mediated gene silencing for the treatment of this devastating disease. Public Health Relevance: The proposed studies are designed to develop a versatile tool that can be used to validate potential therapeutic targets for human neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), using mouse models of disease. The specific experiments described in this proposal will help to identify a therapeutic window in the disease course during which treatments for ALS would be most likely to succeed. These studies will provide critical data to aid in the development of effective therapies for familial ALS and will yield a novel reagent that can be readily adapted for use in future target validation efforts.